Reagents and methods for replication, transcription, and translation in semi-synthetic organisms

Abstract

Disclosed herein are compositions, methods, cells, engineered microorganisms, and kits for increasing the production of proteins or polypeptides comprising one or more unnatural amino acids. Further provided are compositions, cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.

1 - 11 . (canceled) 
     
     
         12 . A plasmid comprising a gene encoding a transfer RNA (tRNA) and/or a gene encoding a protein of interest, wherein the gene comprises at least one nucleobase of the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         each X is independently carbon or nitrogen: 
         R 2  is present when X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide group: 
         Y is sulfur, oxygen, selenium, or secondary amine; and 
         E is oxygen, sulfur, or selenium: 
         or TPT3 
       
       
         
           
           
               
               
           
         
          and at least one complementary base-pairing nucleobase selected from 
       
       
         
           
           
               
               
           
         
          wherein the complementary base-pairing nucleobase is in a complementary base-pairing site. 
       
     
     
         13 . An mRNA encoded by the plasmid of  claim 12  encoding the tRNA or the protein. 
     
     
         14 . (canceled) 
     
     
         15 . A transfer RNA (tRNA) comprising a nucleobase of the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         each X is independently carbon or nitrogen: 
         R 2  is present when X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide group: 
         Y is sulfur, oxygen, selenium, or secondary amine; and 
         E is oxygen, sulfur, or selenium, 
         the tRNA comprising:
 an anticodon, wherein the anticodon comprises the nucleobase, optionally wherein the nucleobase is at the first, second, or third position of the anticodon; and 
 a recognition element, wherein the recognition element promotes selective charging of the tRNA with an unnatural amino acid by an aminoacyl tRNA synthetase. 
 
       
     
     
         16 - 18 . (canceled) 
     
     
         19 . A structure comprising the formula:
   N1-Zx-N2   wherein:   each Z is independently a nucleobase of the structure:   
       
         
           
           
               
               
           
         
         wherein: 
         each X is independently carbon or nitrogen: 
         R 2  is present when X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide group: 
         Y is sulfur, oxygen, selenium, or secondary amine; and 
         E is oxygen, sulfur, or selenium, 
         which is bonded to a ribosyl or deoxyribosyl or analog thereof, 
         N1 is one or more nucleotides or analogs thereof, or a terminal phosphate group attached at the 5′ end of the ribosyl or deoxyribosyl or analog thereof of Z; 
         N2 is one or more nucleotides or analogs thereof, or a terminal hydroxyl group attached to the 3′ end of the ribosyl or deoxyribosyl or analog thereof of Z; and 
         x is an integer from 1 to 20. 
       
     
     
         20 - 26 . (canceled) 
     
     
         27 . A method of transcribing a DNA to a tRNA or an mRNA encoding a protein comprising:
 contacting a DNA comprising a gene encoding the tRNA or protein with ribonucleoside triphosphates and an RNA polymerase, wherein the gene encoding the tRNA or protein comprises a first unnatural base paired to and forming a first unnatural base pair with a second unnatural base, and wherein the ribonucleoside triphosphates comprise a third unnatural base capable of forming a second unnatural base pair with the first unnatural base, wherein the first unnatural base pair and the second unnatural base pair are not the same.   
     
     
         28 . The method of  claim 27 , wherein the ribonucleoside triphosphates further comprise a fourth unnatural base, wherein the fourth unnatural base is capable of forming a third unnatural base pair with the third unnatural base. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 28 , further comprising, before contacting the DNA with the ribonucleoside triphosphates and RNA polymerase, replicating the DNA by contacting the DNA with deoxyribonucleoside triphosphates and a DNA polymerase, wherein the ribonucleoside triphosphates comprise a fifth unnatural base capable of forming a fourth unnatural base pair with the first unnatural base, wherein the first unnatural base pair and the fourth unnatural base pair are not the same. 
     
     
         31 . The method of  claim 28 , wherein the first unnatural base comprises TPT3, the second unnatural base comprises CNMO or NaM, the third unnatural base comprises TAT1, and the fourth unnatural bases comprises NaM or 5FM. 
     
     
         32 . The method of  claim 27 , wherein the method comprises use of a semi-synthetic organism, optionally wherein the organism is a bacterium, optionally wherein the bacterium is  Escherichia coli.    
     
     
         33 . The method of  claim 28 , wherein at least one of the first unnatural base, the second unnatural base, the third unnatural base, or the fourth unnatural base comprises: 
       
         
           
           
               
               
           
         
       
     
     
         34 - 36 . (canceled) 
     
     
         37 . The method of  claim 33 , wherein at least one of the first unnatural base, the second unnatural base, the third unnatural base, or the fourth unnatural base is 
       
         
           
           
               
               
           
         
       
     
     
         38 - 43 . (canceled) 
     
     
         44 . The method of claim  34 , wherein:
 (i) the first unnatural base is   
       
         
           
           
               
               
           
         
          the second unnatural base is 
       
       
         
           
           
               
               
           
         
          the third unnatural base is 
       
       
         
           
           
               
               
           
         
          and the fourth unnatural base is 
       
       
         
           
           
               
               
           
         
          the 
         (ii) the first unnatural base is 
       
       
         
           
           
               
               
           
         
          the second unnatural base is 
       
       
         
           
           
               
               
           
         
          the third unnatural base is 
       
       
         
           
           
               
               
           
         
          and the fourth unnatural base is 
       
       
         
           
           
               
               
           
         
         (iii) the first unnatural base is 
       
       
         
           
           
               
               
           
         
          the second unnatural base is 
       
       
         
           
           
               
               
           
         
          the third unnatural base is 
       
       
         
           
           
               
               
           
         
          and the fourth unnatural base is 
       
       
         
           
           
               
               
           
         
          or 
         (iv) the first unnatural base is 
       
       
         
           
           
               
               
           
         
          the second unnatural base is 
       
       
         
           
           
               
               
           
         
          the third unnatural base is 
       
       
         
           
           
               
               
           
         
          and the fourth unnatural base is 
       
       
         
           
           
               
               
           
         
       
     
     
         45 - 53 . (canceled) 
     
     
         54 . The method of  claim 27 , wherein the mRNA is transcribed, and the method further comprises translating the mRNA into a protein, wherein the protein comprises an unnatural amino acid at a position corresponding to a codon of the mRNA that comprises the third unnatural base. 
     
     
         55 - 58 . (canceled) 
     
     
         59 . The method of  claim 54 , wherein the at least one unnatural amino acid:
 is a lysine analogue;   comprises an aromatic side chain;   comprises an azido group;   comprises an alkyne group; or   comprises an aldehyde or ketone group.   
     
     
         60 . (canceled) 
     
     
         61 . The method of  claim 59 , wherein the at least one unnatural amino acid comprises N6-azidoethoxy-carbonyl-L-lysine (AzK) or N6-propargylethoxy-carbonyl-L-lysine (PraK). 
     
     
         62 - 83 . (canceled) 
     
     
         84 . A method of transcribing DNA, the method comprising:
 providing one or more DNAs comprising (1) a gene encoding a protein, wherein a template strand of the gene encoding the protein comprises a first unnatural base and (2) a gene encoding a tRNA, wherein a template strand of the gene encoding the tRNA comprises a second unnatural base capable of forming a base pair with the first unnatural base;   transcribing the gene encoding the protein to incorporate a third unnatural base into an mRNA, the third unnatural base capable of forming a first unnatural base pair with the first unnatural base;   transcribing the gene encoding a tRNA to incorporate a fourth unnatural base into a tRNA, wherein the fourth unnatural base is capable of forming a second unnatural base pair with the second unnatural base, wherein the first unnatural base pair and the second unnatural base pair are not the same.   
     
     
         85 - 102 . (canceled)